Progress in understanding virus entry has been dominated by pH-dependent viruses, principally because entry can be synchronized by lowering pH and quantitative assays exist to measure this en masse fusion event. Much less attention has been paid to pH-independent viruses such as retroviruses. One of the most important unresolved issues in this field is where retroviruses penetrate the cell membrane and if cellular cues, other than receptor interaction, are required to trigger entry. To identify factors important for Murine leukemia virus entry we designed and screened a custom siRNA library targeting genes important for endocytosis and receptor trafficking. We compared infection of Friend murine leukemia virus (Fr-MLV) to Vesicular stomatitis virus, Venezuelan equine encephalitis virus and Ebola using an envelope pseudotyping system. Genes corresponding to the Rac1-PAK1-LIMK1 pathway of actin regulation were identified as key for infection by Fr-MLV but not the other viruses. Also, Dynamin, EEA1 and Eps15R were identified as important. The latter 3 genes play important roles in endocytosis. Since the only difference between the pseudotypes is the source of envelope protein used, it is likely that the differences seen were due to differences in entry pathway. In this proposal we will test the role of the actin and endocytosis for Murine leukemia virus entry. The role of actin may be for trafficking of receptor across the cell surface or endocytosis itself. We have developed a new virus entry assay that measures virus entry kinetics in real time. The assay provides the highest possible detail for entry measurements. This assay allows us to define the role of each gene for entry and permits us to distinguish roles in trafficking or endocytosis. This study will provide new insight into the entry process of retroviruses including HIV and enveloped viruses in general. Knowledge of the entry pathway will in turn aid in development of drugs that will block this first step in infection and prevent cell to cell spread of virus. [unreadable] [unreadable] [unreadable]